两种甘蔗杆状病毒SCBIMV和SCBMOV实时荧光定量PCR检测方法的建立及应用

发布时间：2023-02-06 17:00

　　甘蔗杆状病毒(Sugarcane bacilliform viruses,SCBVs)属于花椰菜花叶病毒科Caulimoviridae杆状DNA病毒属Badnavirus,是危害甘蔗的主要病毒之一,在世界各主要甘蔗产区均有发现,严重影响甘蔗的产量和品质。因此,急需建立一种快速、灵敏、特异的检测方法,为阻止SCBV病毒跨区域传播,为该病害隔离检疫和健康脱毒种苗质量监控提供关键技术支撑。本研究针对2012年国际病毒分类委员会ICTV公布的两个SCBV病毒种Sugarcane bacilliform IM virus(SCBIMV,NC₀03031)和Sugarcane bacilliform MO virus(SCBMOV,NC₀08017)的反转录酶/RNA 酶 H(RT/RNase H)序列分别设计了一套特异性引物和探针,通过优化反应体系和条件,建立了灵敏度高、特异性强、重现性好的TaqMan实时荧光定量PCR(qPCR)检测方法。该方法以重组质粒PMD18T-IM(SCBIMV)和pSCBV20(SCBMOV)重组质粒DNA为模板(109-...

【文章页数】：87 页

【学位级别】：硕士

【文章目录】：
摘要 Abstract 1 Introduction
1.1 Sugarcane production in China
1.2 Sugarcane viral diseases in China
    1.2.1 Overview of viral diseases
    
1.2.1.1 Viral diseases controls
    
1.2.1.2 Usages of disease-resistance cultivars
    
1.2.1.3 Tissue culture
    
1.2.1.4 Transgenic plants
1.3 Sugarcane bacilliform virus
    1.3.1 Geographical distribution
    1.3.2 Symptoms
    1.3.3 Host range
    1.3.4 Transmission
    1.3.5 Genome characteristics
    1.3.6 Variability and Molecular identification
    1.3.7 Detection methods of SCBV and their limitations
    
1.3.7.1 Symptomatology
    
1.3.7.2 Electron microscopy
    
1.3.7.3 Serology
    
1.3.7.4 Polymerase chain reaction (PCR)
    
1.3.7.5 Immunocapture PCR (IC-PCR)
1.4 SCBV disease control
1.5 Purpose and objective of study 2 Materials and Methods
2.1 Leaves sampling
2.2 Reagents and kits
2.3 DNA Isolation and purification
2.4 Primer and probe design
2.5 Plasmids generation
2.6 Plasmid extraction and dilution
2.7 qPCR assay
2.8 qPCR standard curve construction
2.9 Sensitivity test of the qPCR
2.10 Specificity test of the qPCR
2.11 Conventional PCR
2.12 PCR products purification
2.13 PCR products cloning and sequencing
2.14 Field samples detection for SCBV
2.15 Sequence alignment
2.16 Phylogenetic analysis
2.17 Sequence identity and genetic distance analysis 3 Results
3.1 Primer specificity analysis in-silico
3.2 Efficiency of Real-Time qPCR Assay
3.3 Sensitivity of the qPCR
3.4 Specificity of the qPCR
3.5 Field samples detection for SCBV
3.6 SCBV genotypes identification
    3.6.1 Phylogenetic grouping
    3.6.2 Sequences identity
    3.6.3 Sequence genetic distance 4 Discussion and Conclusion
4.1 qPCR assays is more sensitive and efficient for SCBV detection
4.2 Highly genetic variation among the SCBV genotypes/strains
4.3 Quarantine inspection is necessary in plant cutting exchange
4.4 Conclusions References Published Papers Appendices Acknowledgement



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